Nucleic acid agents for downregulating H19 and methods of using same

ABSTRACT

The present invention provides isolated oligonucleotides capable of down-regulating a level of H19 mRNA in cancer cells. Articles of manufacture comprising agents capable of downregulating H19 mRNA in combination with an additional anti-cancer treatment are disclosed as well as methods of treating cancer by administering same.

This application is a 371 filing of International Patent ApplicationPCT/IL2006/000785, filed Jul. 6, 2006, which claims the benefit of U.S.application No. 60/696,795 filed Jul. 7, 2005.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to nucleic acid agents for downregulatingH19 and use thereof for the treatment of cancer.

H19 was the first human imprinted non protein-coding gene to beidentified showing expression of only the maternal allele. It is alsoimprinted in mice. H19 was mapped on the short arm of the humanchromosome 11, band 15.5, homologous to a region of murine chromosome 7.It belongs to a group of genes that very likely does not code for aprotein product. H19 gene is abundantly expressed in embryogenesis butis shut off in most tissues after birth. However, studies of varioustumors have demonstrated a re-expression or an over-expression of theH19 gene when compared to healthy tissues. Moreover in cancers ofdifferent etiologies and lineages, aberrant expression in allelicpattern was observed in some cases. While H19 shows mono-allelicexpression in most tissues throughout development, with the exception ofgerm cells at certain stages of maturation, and in extra-villoustrophoblasts, bi-allelic expression of this gene, referred as“relaxation of imprinting” or “loss of imprinting” (LOI), have beenfound in an increasing number of cancers, for example, hepatocellularcarcinoma, liver neoplasms of albumin SV40 T antigen-transgenic rats,lung adenocarcinoma, esophageal, ovarian, rhabdomyosarcoma, cervical,bladder, head and neck squamous cell carcinoma, colorectal, uterus andin testicular germ cell tumors. Today nearly 30 types of cancers showdysregulated expression of H19 gene as compared to healthy tissues, withor without LOI. For a recent review see Matouk et al (Matouk et al,2005, Gene Ther Mol Biol).

It was also shown that H19 over-expression of ectopic origin conferred aproliferative advantage for breast epithelial cells in a soft agar assayand in several combined immunodeficient (SCID) mice (Lottin et al, 2002,Oncogene 21, 1625-1631). In tumors formed by the injection of cells of achoriocarcinoma-derived cell line (JEG-3), and a bladder carcinoma cellline (T24P), the H19 level is very high when compared to the level ofH19 in cells prior to injection [Rachmilewitz et al, 1995, Oncogene 11,863-870].

Moreover, certain known carcinogens upregulate the expression of the H19gene. A dramatic elevation of H19 RNA levels was detected in the airwayepithelium of smokers without (LOI) [Kaplan et al, 2003, Cancer Res 63,1475-1482]. BBN (a known carcinogen of the bladder) also induces theexpression of H19 gene in the rat model of bladder cancer [Ariel et al,2004, Mol Carcinog 41, 69-76]. Likewise, Diethylnitrosamine (a knowncarcinogen of the liver) induces the expression of H19 in a mice modelof hepatocellular carcinoma [Graveel et al, 2001, Oncogene 20,2704-2712]. All of these observations and others contradict the initialproposal that H19 is a tumor suppressor gene.

Comparing patterns of gene expression in two homogeneous cellpopulations that only differ in the presence or absence of H19 RNA haveidentified plenty of downstream effectors of H19 RNA, among these aregroup of genes that were previously reported to play crucial roles insome aspects of the tumorigenic process. H19 RNA presence may enhancethe invasive, migratory and angiogenic capacity of the cell by upregulating genes that function in those pathways, and could thuscontribute at least to the initial steps of the metastatic cascade.Additional studies highlight H19's potential role in promoting cancerprogression and tumor metastasis by being a responsive gene to HGF/SF.

The specific expression of H19 gene in cancer cells has prompted its usein clinical applications for diagnosing cancer.

Thus, U.S. Pat. No. 5,955,273 to the present inventors teaches the useof H19 gene as a tumor specific marker.

PCT Pub. No. WO 9524503 teaches the detection of malignancies and theirgrading with a H19 gene probe by in-situ hybridization—useful fordetecting presence/absence of malignancy in pediatric Wilms' Tumor.

PCT Pub. No. WO 0403159 teaches down-regulation of H19 for treatingdiseases associated with angiogenesis, such as cancer. However,down-regulation of H19 was not shown to reduce tumor size or volume,neither was specific and efficacious siRNA agents capable ofdown-regulating H19 taught. Furthermore, PCT Pub. No. WO 0403159 doesnot teach use of anti H19 agents as part of a combination therapy fortreating cancer.

There is thus a widely recognized need for, and it would be highlyadvantageous to have, methods and compositions for down-regulating H19for cancer treatment.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided anisolated oligonucleotide selected from the group consisting of SEQ IDNO: 1, 2, 3 and 4.

According to another aspect of the present invention there is provided apharmaceutical composition comprising, a pharmaceutically acceptablecarrier and as an active ingredient at least one isolatedoligonucleotide selected from the group consisting of SEQ ID NO: 1, 2, 3and 4.

According to yet another aspect of the present invention there isprovided a use of an isolated oligonucleotide selected from the groupconsisting of SEQ ID NO: 1, 2, 3 and 4 for the preparation of amedicament for the treatment of cancer.

According to still another aspect of the present invention there isprovided a method of treating cancer comprising:

(a) administering to, or expressing in cells of a subject in needthereof a therapeutically effective amount of an agent capable ofdown-regulating a level and/or activity of H19 mRNA, and

(b) providing to the subject a cancer therapy, thereby treating cancer.

According to an additional aspect of the present invention there isprovided a use of an agent capable of down-regulating a level and/oractivity of H19 mRNA for the preparation of a medicament for thetreatment of cancer in combination with a cancer therapy.

According to yet an additional aspect of the present invention there isprovided a method of treating cancer comprising administering to, orexpressing in cells of a subject in need thereof a therapeuticallyeffective amount of at least one oligonucleotide selected from the groupconsisting of SEQ ID NO: 1, 2, 3 and 4, thereby treating cancer.

According to still an additional aspect of the present invention thereis provided an article of manufacture comprising an agent capable ofdownregulating a level and/or activity of H19 mRNA and an additionalanti cancer agent identified for treating cancer.

According to further features in preferred embodiments of the inventiondescribed below, the agent capable of downregulating a level and/oractivity of H19 mRNA is a nucleic acid agent.

According to still further features in the described preferredembodiments, the nucleic acid agent is selected from the groupconsisting of:

(a) a single stranded polynucleotide for inhibiting the transcription ofthe H19 RNA from the H19 gene;

(b) a single stranded polynucleotide for hybridizing to the H19 mRNAthereby leading to a reduction of H19 mRNA activity;

(c) a double stranded polynucleotide, leading to degradation of the H19mRNA;

(d) a triplex forming polynucleotide for cleaving the H19 mRNA;

(e) a catalytic polynucleotide for cleaving the H19 mRNA;

(f) a single stranded polynucleotide for hybridizing to the H19 mRNAleading to enzymatic degradation thereof; and

(g) nucleic acid sequences coding for any one of (a) to (f).

According to still further features in the described preferredembodiments, the nucleic acid agent is selected from the groupconsisting of an siRNA, a ribozyme and a DNAzyme.

According to still further features in the described preferredembodiments, the nucleic acid agent is a siRNA.

According to still further features in the described preferredembodiments, the siRNA comprises a nucleic acid sequence selected fromthe group consisting of SEQ ID NO: 1-4.

According to still further features in the described preferredembodiments, the administering is effected in situ.

According to still further features in the described preferredembodiments, the cancer is selected from the group consisting ofpediatric solid tumors, Wilms' tumor, Hepatoblastoma, Embryonalrhabdomyosarcoma, Germ cell tumors and trophoblastic tumors, testiculargerm cells tumors, immature teratoma of ovary, sacrococcygeal tumors,Choriocarcinoma, Placental site trophoblastic tumors, Epithelial adulttumors, Bladder carcinoma, Hepatocellular carcinoma, Ovarian carcinoma,Cervical carcinoma, Lung carcinoma, Breast carcinoma, Squamous cellcarcinoma in head and neck, Esophageal carcinoma, Neurogenic tumor,Astrocytoma, Ganglioblastoma, Neuroblastoma.

According to still further features in the described preferredembodiments, the cancer is a bladder carcinoma or a hepatocellularcarcinoma.

According to still further features in the described preferredembodiments, the agent capable of downregulating a level and/or activityof H19 mRNA is co-formulated with the additional anti cancer agent.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing nucleotide agents capable ofdown-regulating H19 RNA both alone and in combination for the treatmentof cancer.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIGS. 1A-F are photographs and schematic diagrams illustrating thepresence of an alternative splice variant of H19 in human embryonic andplacental specimens. FIG. 1A is a schematic representation of chromosome11 showing the location of the H19 gene, which is composed of 5 exons(E1-5) (solid boxes) and 4 short introns (lines between boxes). Theposition of primers used in the PCR reaction (SEQ ID NOs: 8 and 9) aremarked by horizontal arrows and are 117 and 816 bases downstream to thetranscription initiation site. The spliced segment lacking is indicatedby a grey box. FIGS. 1B-D are photographs of ethidium bromide stainedgel for RT-PCR reaction. The following cells were analyzed: Hep3B andSKHep1 (hepatocellular carcinomas cell lines); RT4 and Umuc3 (bladdercarcinoma cell lines); placental specimens: 1^(st) trim (firsttrimester); Mola, hydatiform mole; 3^(rd) trim (third trimester); LG-BC,low grade bladder cancer; HG-BC, high-grade bladder cancer; NB, normalbladder; NC, normal colon; CClym, colon cancer metastasized to lymphnode; CCliv colon cancer metastasized to liver; CC, colon cancer; InFIGS. 1B-D, the lanes marked by C refer to a negative control; M refersto marker 100 bp ladder. The sizes of the products are indicated on theright. FIG. 1E is a photograph of an RNase protection assay. Arrowheadindicates the presence of the alternative splice variant 344 bases inthird trimester placental tissue. Two other excessive additional bands,which could indicate the presence of another alternative splice variant,which were undetectable by RT-PCR reaction, were also detected. FIG. 1Fis a partial sequence analysis (SEQ ID NO: 14) of the alternativespliced variant revealing a skipping region of 366 bases from exon 1.The underlined sequence indicates the splice junction. (Nucleotidenumbering begins at the start codon).

FIGS. 2A-B are photographs of ethidium bromide stained gels illustratingthe effect of increasing concentrations of CoCl₂ on the expression levelof H19 RNA in Hep3B cells. FIG. 2A illustrates the RT-PCR products ofthe H19 gene in Hep3B cells. FIG. 2B illustrates the RT-PCR products ofthe GADPH gene as a positive control for RT-PCR integrity in Hep3B cellsFor FIGS. 2A and 2B, untreated Hep3B (lane1); 50, 100, 200, 300 and 400μM CoCl₂ treated cells (lanes 2, 3, 4, 5 and 6) respectively.

FIGS. 3A-F are bar graphs and photographs illustrating the effectivenessof in-vitro down-regulation of H19 using the siRNAs of the presentinvention. FIGS. 3A-C are ethidium bromide stained gels illustrating theeffect of different H19 siRNA duplexes on the expression level of H19 ina Hep3B cell line under normal culture condition (FIG. 3A) and hypoxiamimicking condition (FIG. 3B) as tested by RT-PCR analysis. FIG. 3Aillustrates Hep3B cells transfected with unrelated siRNA duplex thattargets luciferase gene (lane 1) and with the four H19 siRNA duplexes(Lanes 2-5) (SEQ ID NOs. 1-4) and their equimolar mixtures (lane 6), andlipofectamine 2000 without siRNA (Mock) (Lane 7). Note, all siRNA agentstested (SEQ ID NOs: 1-4) were at least 50% effective in reducing themRNA level of H19. C=PCR blank. FIGS. 3B and 3C illustrate Hep3B cellstransfected in normal medium with siRNA duplex that targets luciferasegene—(SEQ ID NO: 5) (Lanes 1 and 5) and with 3 different H19 siRNAduplexes (SEQ ID NOs. 1, 3, and 4) (lanes 2-4). 24 hour posttransfection, media was changed and 100 μM CoCl2 containing media wasadded except for lane 5 which shows cells which continued to grow innormal culture media. The incubation was for a further 22 hours. RT-PCRproducts are shown for both H19 (FIG. 3B) and GADPH (FIG. 3C) genes as apositive control for RT-PCR integrity.

FIGS. 3D-E are ethidium bromide stained gels illustrating the effect ofH19 siRNA duplexes (SEQ ID NO:1) on the expression level of H19 in aUMUC3 cell line under normal culturing conditions and hypoxic conditionsas tested by RT-PCR analysis. For FIGS. 3F and 3G, GFP siRNA transfectedUMUC3 cells (lane 1), plus H19 siRNA—SEQ ID NO: 1 (lane 2) in normoxicconditions, and GFP siRNA transfected UMUC3 cells (lane 3), plus H19siRNA (lane 4) in hypoxic conditions respectively.

FIG. 3F is a bar graph illustrating the reduction in colony numbersfollowing hypoxia recovery following H19 siRNA (SEQ ID NO:3)transfection in Hep3B cells as compared to GFP siRNA control treatedcells.

FIGS. 4A-D are bar graphs and photographs illustrating that transientH19 RNA downregulation in Hep3B cells inhibits tumorigenicity in vivo.Hep3B cells were transiently transfected with H19 siRNA 3 (SEQ ID NO: 3)or anti-Luc siRNA (SEQ ID NO: 5). Forty eight hours post transfection,cells were washed twice with PBS, trypsinized and counted. 1.5×10⁶ cellsreceiving anti-H19 siRNA and anti-Luc siRNA were injected subcutaneouslyinto the dorsal part of CD-1 nude mice (n=7 for both, and 4 for mocktransfected). Palpable tumors were observed 15 days post inoculation inmice inoculated with Hep3B, transiently transfected with anti-Luc siRNA.Tumor volumes were followed up and measured using a caliper until day 30post inoculation, after which mice were sacrificed. Significant (p<0.03)reductions of about 82% of both mean tumor weights (A) (±standard error)and mean tumor volumes (p<0.03) (B) (±standard error) were observed.Values represent end-points just prior to and following sacrificinganimals. Shown are also representative features of tumors in 2 mice ofeach group (mice 1 and 2 are the H19 siRNA3 (SEQ ID NO: 3) treatedanimals, and mice 3 and 4 are the anti-Luc siRNA (SEQ ID NO: 5) treatedanimals) prior to tumor surgical exposure (C), and following exposure oftheir internal tumors (D).

FIGS. 5A-D are bar graphs and photographs illustrating the in vivoeffect of siRNA-H19 on human bladder carcinoma cells-UMUC3. One millionUMUC3 cells were injected subcutaneously to athymic mice (n=3 for GFPsiRNA (SEQ ID NO; 6), and 5 for siRNA H19 (SEQ ID No:1), 48 hours aftertransiently transfected with siRNAs. Palable tumors were observed 6weeks later in 2 out of 3 mice receiving UMUC3 transiently transfectedwith anti-GFP-siRNA, while in none of those receiving siRNA H19 (n=5).Mice were sacrificed 8 week after inoculation. Mean tumor volumes (B,P<0.05), and mean tumor weights (A, p<0.06) are depicted. Valuesrepresent end-points just before and after sacrificing animals. Picturesdepict the external features of the tumors in mice inoculated with UMUC3transfected with anti-GFP siRNA (C), and siRNA H19 (D).

FIG. 6 is a bar graph illustrating the effect of H19 siRNA (SEQ ID NO:3) transfection in Hep3B cells on proliferation under normal cultureconditions.

FIGS. 7A-D are bar and line graphs illustrating the effect ofintratumoral administration of H19 siRNAs (SEQ ID No 1; SEQ ID NO: 3) oranti GFP siRNA (SEQ ID NO: 6) on previously injected human bladdercarcinoma cells-UMUC3 (FIGS. 7A-B) SEQ ID NO: 1 and Hep3B cells SEQ IDNO: 3 (FIGS. 7C-D) in CD-1 nude mice. FIG. 7A is a line graph depictingthe change in tumor volume over time following injection of siRNA-H19(SEQ ID NO: 1) or anti GFP siRNA into UMUC-3 treated mice. FIG. 7B is abar graph depicting the change in tumor weight following injection ofsiRNA-H19 or anti GFP siRNA into UMUC-3 treated mice. FIG. 7C is a bargraph depicting tumor volume following injection of siRNA-H19 or antiGFP siRNA into Hep3B-treated mice. FIG. 7D is a bar graph depicting thechange in tumor weight following injection of siRNA-H19 (SEQ ID NO: 3)or anti GFP siRNA into Hep3B treated mice.

FIGS. 8A-D are bar graphs and photographs illustrating the effect of H19ectopic expression on the growth of the human bladder carcinoma cellsTA11 (negative for H19) and TA31 (high expresser of H19) in vivo: Equalamounts (2×10⁶) of TA31H19-high and TA11H19-ve cells were implantedsubcutaneously to CD-1 mice (n=5, each). Two weeks later, palable tumorsappeared and tumor volumes were measured for additional two weeks usinga caliper. Shown are end point measurements of the mean tumor volumes ofthe two groups (FIG. 8A), their mean tumor volumes kinetics (FIG. 8B),and a representative gross morphology of tumors derived from theTA11H19-ve (FIG. 8C) and TA31H19-high cells (FIG. 8D).

FIGS. 9A-C are photographs illustrating that H19 RNA is induced byhypoxic stress in Hep3B cell line and that siRNA directed against H19very efficiently impedes its induction. Hep3B cells were seeded andtransfected either with anti H19 siRNA or anti luc-siRNA. Twenty fourhours post transfection, cells were either placed into an Aneoropackrectangular jar (Mitsubishi Chemical Company, Japan) to create ahypoxic-like condition within an hour, or left under normal oxygenconcentration. Incubation lasted for 24 hours prior to RNA extraction.Shown are RT-PCR analyses for H19 RNA. FIG. 9A: Hep3B transfected withanti-luc siRNA (SEQ ID NO: 5) (lanes 1, 2) and anti H19 siRNA (SEQ IDNO: 3) (lanes 3, 4) both in normal (lanes 1, 3) and hypoxic (lanes 2, 4)culture conditions, respectively. PCR analysis of a house-keeping gene,GAPDH, (FIG. 9B), and uPAR (FIG. 9C).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to nucleic acid agents for downregulatingthe level and/or activity of H19 and pharmaceutical compositions andmethods of using same. Specifically, the present invention relates tomethods and compositions for treating cancer.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

H19 is an imprinted gene that demonstrates maternal monoallelicexpression, and very likely does not code for a protein. It isabundantly expressed during embryogenesis and fetal development, but istypically shut off after birth in most tissues. However in an increasingnumbers of cancers of different origins, expression of H19 RNA isup-regulated and an aberrant allelic pattern of expression was observedin some cases, suggesting that H19 may play a role in tumorigenesis.

While reducing the present method to practice, the present inventorsdesigned through laborious bioinformatics modeling specific siRNAs whichcan effectively down-regulate H19 mRNA. The siRNAs of the presentinvention were selected using four different search engines to ensurethat the optimal siRNAs were chosen.

As illustrated in FIG. 3A all the siRNAs that were generated were foundto be efficient in down-regulating H19 mRNA. Furthermore, the presentinventors showed that these siRNAs were able to down-regulate H19 mRNAunder both normal and hypoxic conditions (FIGS. 3B-E). This is ofparticular relevance since tumor growth is associated with hypoxia,which in turn is associated with up-regulation of H19 RNA.

The siRNAs of the present invention were able to both prevent tumorformation and even reduce on-going disease by reduction ofpre-established tumor volume and weight.

As illustrated in Example 4, administration of human carcinoma cells(Hep3B and UMUC3), previously transfected with H19 siRNA, into micecaused a very significant lowering in tumor weight (FIG. 4A and FIG. 5A)and volume (FIG. 4B and FIG. 5B) than administration of the identicalcells transfected with control siRNA.

Furthermore, as illustrated in Example 6, injection of H19 siRNAdirectly into tumors induced by UMUC3 cells, caused a very significantreduction of about 90% of mean tumor volumes (FIG. 7A), and of about 88%of mean tumor weights (FIG. 7B).

In Hep3B induced tumors, an approximate 40% reduction of tumor weights(FIG. 7C) and 56% reduction of tumor volumes (FIG. 7D) were observedfollowing administration of H19 siRNA.

Altogether, these results undoubtedly place agents capable ofdown-regulating H19 as realistic candidates for both the prophylacticand therapeutic treatment of cancer.

Additionally, the present invention also envisages using agents capableof down-regulating H19 mRNA in combination therapy. It is wellestablished that solid tumors especially those encountering hypoxicregions are resistant to cancer therapy. It is anticipated by thepresent invention anti H19 agents may act to sensitize a patient to apre-established cancer therapy (e.g., radio-therapy, chemotherapy).

Thus according to one aspect of the present invention, there is providedan article of manufacture comprising an agent capable of downregulatinga level and/or activity of H19 mRNA and an additional anti cancer agentidentified for the treatment of cancer.

As used herein the term “treating” refers to preventing, alleviating ordiminishing a symptom associated with a cancerous disease. Preferably,treating cures, e.g., substantially eliminates, the symptoms associatedwith cancer.

Any cancer which expresses H19 may be treated according to this aspectof the present invention. Preferable tumors treated according to themethod of the present invention are those which express H19 mRNA duringtumor onset or progression. Such tumors include, but are not limited to,Pediatric solid tumors, Wilms' tumor, Hepatoblastoma, Embryonalrhabdomyosarcoma, Germ cell tumors and trophoblastic tumors, testiculargerm cells tumors, immature teratoma of ovary, sacrococcygeal tumors,Choriocarcinoma, Placental site trophoblastic tumors, Epithelial adulttumors, Bladder carcinoma, Hepatocellular carcinoma, Ovarian carcinoma,Cervical carcinoma, Lung carcinoma, Breast carcinoma, Squamous cellcarcinoma in head and neck, Esophageal carcinoma, Neurogenic tumor,Astrocytoma, Ganglioblastoma, Neuroblastoma. Preferably the tumor is abladder carcinoma or a hepatocellular carcinoma.

As used herein the term “subject” refers to any (e.g., mammalian)subject, preferably a human subject.

As used herein the phrase “H19 mRNA” refers to a transcriptional productof the H19 gene (GenBank Accession No. M32053—SEQ ID NO: 7).

The present inventors have identified a novel splice isoform of theH19RNA gene which is specifically expressed in embryonic tissues and notin carcinoma cells as demonstrated by RT-PCR analysis (FIGS. 1B-D) andRNase protection assay (FIG. 1E). This novel splice isoform was shown tolack part of exon-1 extending from nucleotide 252 to 588 of thetranscription start site as compared to the known H19 transcript as setforth in SEQ ID NO: 7. Accordingly, the H19 RNA of this aspect of thepresent invention preferably comprises exon 1 of the H19 transcript andeven more preferably comprises the RNA sequence denoted by nucleic acidsequence coordinates 252 to 588 of SEQ ID NO: 7.

Since H19 does not encode for a protein, downregulating a level oractivity of H19mRNA is preferably effected at the RNA level.

Preferably the level and/or activity of H19 which is down-regulated isgreater than 10%, more preferably greater than 20%, more preferablygreater than 40%, more preferably greater than 60%, more preferablygreater than 80%, and even more preferably 100%.

Preferably the agent is a nucleic acid agent. More preferably the agentis an oligonucleotide, most preferably a double strandedoligonucleotide.

The decrease in the level of the H19 mRNA may be achieved by severalmechanisms: by inhibiting transcription from the H19 gene to H19 RNA; byinhibition of the maturation process from hnRNA to mRNA; by promotion ofmRNA degradation in the cytoplasm by enzymes (by forming RNA duplexes ortriplexes, and by catalytic cleavage of nucleic acid based enzymes(DNAzymes and RNAzymes).

Thus the anti-H19 mRNA agent in accordance with the invention may beselected from the following:

-   -   1) a single stranded nucleic acid sequence for steric inhibition        of the transcription of H19 RNA from its gene;    -   2) a single stranded nucleic acid sequence for hybridization        with the H19 RNA leading to enzymatic degradation (for example        by RNAses);    -   3) a double stranded nucleic acid sequence, that leads to        degradation of the H19 (by forming siRNA);    -   4) a catalytic nucleic acid sequence for cleavage of the H19        mRNA;    -   5) a triplex forming nucleotide;    -   6) a single stranded nucleic acid sequence for hybridizing the        H19 mRNA thereby leading to a reduction of H19 mRNA activity;        and    -   7) nucleic acid sequences coding for any one of (1) to (6).

According to one embodiment of this aspect of the present invention theagent is a nucleic acid agent comprising a nucleic acid sequence capableof specifically hybridizing (e.g., in cells under physiologicalconditions) to the H19 RNA of the present invention, as described above.

As used herein, the term “nucleic acid agent” refers to asingle-stranded or double-stranded oligomer or polymer of ribonucleicacid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This termincludes polynucleotides composed of naturally occurring bases, sugars,and covalent internucleoside linkages (e.g., backbone), as well aspolynucleotides having non-naturally occurring portions, which functionsimilarly to respective naturally occurring portions.

As used herein, the phrase “capable of hybridizing” refers tobase-pairing, where at least one strand of the nucleic acid agent is atleast partly homologous to H19 mRNA.

Preferably, the nucleic acid agents of the present inventionspecifically hybridize with H19 RNA of the present invention i.e. haveat least a 5 fold preference for hybridizing with H19 RNA as opposed toa non-related RNA molecule (e.g. GAPDH).

The nucleic acid agents designed according to the teachings of thepresent invention can be generated according to any nucleic acidsynthesis method known in the art, including both enzymatic syntheses orsolid-phase syntheses. Equipment and reagents for executing solid-phasesynthesis are commercially available from, for example, AppliedBiosystems. Any other means for such synthesis may also be employed; theactual synthesis of the nucleic acid agents is well within thecapabilities of one skilled in the art and can be accomplished viaestablished methodologies as detailed in, for example: Sambrook, J. andRussell, D. W. (2001), “Molecular Cloning: A Laboratory Manual”;Ausubel, R. M. et al., eds. (1994, 1989), “Current Protocols inMolecular Biology,” Volumes I-III, John Wiley & Sons, Baltimore, Md.;Perbal, B. (1988), “A Practical Guide to Molecular Cloning,” John Wiley& Sons, New York; and Gait, M. J., ed. (1984), “OligonucleotideSynthesis”; utilizing solid-phase chemistry, e.g. cyanoethylphosphoramidite followed by deprotection, desalting, and purificationby, for example, an automated trityl-on method or HPLC.

It will be appreciated that nucleic acid agents of the present inventioncan be also generated using an expression vector as is further describedhereinbelow.

Preferably, the nucleic acid agents of the present invention aremodified. Nucleic acid agents can be modified using various methodsknown in the art.

For example, the nucleic acid agents of the present invention maycomprise heterocylic nucleosides consisting of purines and thepyrimidines bases, bonded in a 3′-to-5′ phosphodiester linkage.

Preferably used nucleic acid agents are those modified either inbackbone, internucleoside linkages, or bases, as is broadly describedhereinunder.

Specific examples of preferred nucleic acid agents useful according tothis aspect of the present invention include oligonucleotides orpolynucleotides containing modified backbones or non-naturalinternucleoside linkages. Oligonucleotides or polynucleotides havingmodified backbones include those that retain a phosphorus atom in thebackbone, as disclosed in U.S. Pat. Nos. 4,469,863; 4,476,301;5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302;5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233;5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;5,563,253; 5,571,799; 5,587,361; and 5,625,050.

Preferred modified oligonucleotide backbones include, for example:phosphorothioates; chiral phosphorothioates; phosphorodithioates;phosphotriesters; aminoalkyl phosphotriesters; methyl and other alkylphosphonates, including 3′-alkylene phosphonates and chiralphosphonates; phosphinates; phosphoramidates, including 3′-aminophosphoramidate and aminoalkylphosphoramidates; thionophosphoramidates;thionoalkylphosphonates; thionoalkylphosphotriesters; andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogues ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts, and free acid forms of the above modifications canalso be used.

Alternatively, modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short-chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short-chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene-containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts, as disclosed in U.S. Pat. Nos. 5,034,506;5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562;5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677;5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240;5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360;5,677,437; and 5,677,439.

Other nucleic acid agents which may be used according to the presentinvention are those modified in both sugar and the internucleosidelinkage, i.e., the backbone of the nucleotide units is replaced withnovel groups. The base units are maintained for complementation with theappropriate polynucleotide target. An example of such an oligonucleotidemimetic includes a peptide nucleic acid (PNA). A PNA oligonucleotiderefers to an oligonucleotide where the sugar-backbone is replaced withan amide-containing backbone, in particular an aminoethylglycinebackbone. The bases are retained and are bound directly or indirectly toaza-nitrogen atoms of the amide portion of the backbone. United Statespatents that teach the preparation of PNA compounds include, but are notlimited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262; each ofwhich is herein incorporated by reference. Other backbone modificationswhich may be used in the present invention are disclosed in U.S. Pat.No. 6,303,374.

Nucleic acid agents of the present invention may also include basemodifications or substitutions. As used herein, “unmodified” or“natural” bases include the purine bases adenine (A) and guanine (G) andthe pyrimidine bases thymine (T), cytosine (C), and uracil (U).“Modified” bases include but are not limited to other synthetic andnatural bases, such as: 5-methylcytosine (5-me-C); 5-hydroxymethylcytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl and otheralkyl derivatives of adenine and guanine; 2-propyl and other alkylderivatives of adenine and guanine; 2-thiouracil, 2-thiothymine, and2-thiocytosine; 5-halouracil and cytosine; 5-propynyl uracil andcytosine; 6-azo uracil, cytosine, and thymine; 5-uracil (pseudouracil);4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, andother 8-substituted adenines and guanines; 5-halo, particularly 5-bromo,5-trifluoromethyl, and other 5-substituted uracils and cytosines;7-methylguanine and 7-methyladenine; 8-azaguanine and 8-azaadenine;7-deazaguanine and 7-deazaadenine; and 3-deazaguanine and3-deazaadenine. Additional modified bases include those disclosed in:U.S. Pat. No. 3,687,808; Kroschwitz, J. I., ed. (1990), “The ConciseEncyclopedia Of Polymer Science And Engineering,” pages 858-859, JohnWiley & Sons; Englisch et al. (1991), “Angewandte Chemie,” InternationalEdition, 30, 613; and Sanghvi, Y. S., “Antisense Research andApplications,” Chapter 15, pages 289-302, S. T. Crooke and B. Lebleu,eds., CRC Press, 1993. Such modified bases are particularly useful forincreasing the binding affinity of the oligomeric compounds of theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines,and N-2, N-6, and O-6-substituted purines, including2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S. et al. (1993),“Antisense Research and Applications,” pages 276-278, CRC Press, BocaRaton), and are presently preferred base substitutions, even moreparticularly when combined with 2′-O-methoxyethyl sugar modifications.

The nucleic acid agents of the present invention is of at least 10, atleast 15, or at least 17 bases specifically hybridizable with H19 RNA.As illustrated in Example 1, the siRNAs of the present invention are 19bases with two 3′ overhangs.

It should be appreciated that the present invention also envisagesagents other than nucleic acid agents that are capable ofdown-regulating H19 RNA such as knock-out agents.

A small interfering RNA (siRNA) molecule is an example of an nucleicacid agents agent capable of downregulating H19RNA. RNA interference isa two-step process. During the first step, which is termed theinitiation step, input dsRNA is digested into 21-23 nucleotide (nt)small interfering RNAs (siRNA), probably by the action of Dicer, amember of the RNase III family of dsRNA-specific ribonucleases, whichcleaves dsRNA (introduced directly or via an expressing vector, cassetteor virus) in an ATP-dependent manner. Successive cleavage events degradethe RNA to 19-21 bp duplexes (siRNA), each strand with 2-nucleotide 3′overhangs [Hutvagner and Zamore Curr. Opin. Genetics and Development12:225-232 (2002); and Bernstein Nature 409:363-366 (2001)].

In the effector step, the siRNA duplexes bind to a nuclease complex toform the RNA-induced silencing complex (RISC). An ATP-dependentunwinding of the siRNA duplex is required for activation of the RISC.The active RISC then targets the homologous transcript by base pairinginteractions and cleaves the mRNA into 12 nucleotide fragments from the3′ terminus of the siRNA [Hutvagner and Zamore Curr. Opin. Genetics andDevelopment 12:225-232 (2002); Hammond et al., (2001) Nat. Rev. Gen.2:110-119 (2001); and Sharp Genes. Dev. 15:485-90 (2001)]. Although themechanism of cleavage is still to be elucidated, research indicates thateach RISC contains a single siRNA and an RNase [Hutvagner and ZamoreCurr. Opin. Genetics and Development 12:225-232 (2002)].

It is possible to eliminate the “initiation step” by providing a priorisiRNA.

Because of the remarkable potency of RNAi, an amplification step withinthe RNAi pathway has been suggested. Amplification could occur bycopying of the input dsRNAs, which would generate more siRNAs, or byreplication of the siRNAs formed. Alternatively or additionally,amplification could be effected by multiple turnover events of the RISC[Hammond et al., Nat. Rev. Gen. 2:110-119 (2001), Sharp Genes. Dev.15:485-90 (2001); Hutvagner and Zamore Curr. Opin. Genetics andDevelopment 12:225-232 (2002)]. For more information on RNAi see thefollowing reviews Tuschl ChemBiochem. 2:239-245 (2001); Cullen Nat.Immunol. 3:597-599 (2002); and Brantl Biochem. Biophys. Act. 1575:15-25(2002).

Synthesis of RNAi molecules suitable for use with the present inventioncan be effected as follows. First, the H19 nucleic acid sequence targetis scanned downstream for AA dinucleotide sequences. Occurrence of eachAA and the 3′ adjacent 19 nucleotides is recorded as potential siRNAtarget sites.

Second, potential target sites are compared to an appropriate genomicdatabase (e.g., human, mouse, rat etc.) using any sequence alignmentsoftware, such as the BLAST software available from the NCBI server(www.ncbi.nlm.nih.gov/BLAST/). Putative target sites that exhibitsignificant homology to other coding sequences are filtered out.

Qualifying target sequences are selected as template for siRNAsynthesis. Preferred sequences are those including low G/C content asthese have proven to be more effective in mediating gene silencing ascompared to those with G/C content higher than 55%. Several target sitesare preferably selected along the length of the target gene forevaluation. For better evaluation of the selected siRNAs, a negativecontrol is preferably used in conjunction. Negative control siRNApreferably include the same nucleotide composition as the siRNAs butlack significant homology to the genome. Thus, a scrambled nucleotidesequence of the siRNA is preferably used, provided it does not displayany significant homology to any other gene.

Examples of siRNAs which are capable of down-regulating H19 that may beused according to this aspect of the present invention are those setforth by SEQ ID NOs: 1-4.

Since these molecules were shown effective in reducing tumor size andvolume the present invention also envisages treatment of cancer usingthese molecules alone and not necessarily in combination.

Another agent capable of downregulating the expression of a H19 RNA is aDNAzyme molecule capable of specifically cleaving its encodingpolynucleotide. DNAzymes are single-stranded nucleic acid agents whichare capable of cleaving both single and double stranded target sequences(Breaker, R. R. and Joyce, G. Chemistry and Biology 1995; 2:655;Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 1997; 94:4262).A general model (the “10-23” model) for the DNAzyme has been proposed.“10-23” DNAzymes have a catalytic domain of 15 deoxyribonucleotides,flanked by two substrate-recognition domains of seven to ninedeoxyribonucleotides each. This type of DNAzyme can effectively cleaveits substrate RNA at purine:pyrimidine junctions (Santoro, S. W. &Joyce, G. F. Proc. Natl, Acad. Sci. USA 199; for rev of DNAzymes seeKhachigian, L M [Curr Opin Mol Ther 4:119-21 (2002)].

Examples of construction and amplification of synthetic, engineeredDNAzymes recognizing single and double-stranded target cleavage siteshave been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al. DNAzymesof similar design directed against the human Urokinase receptor wererecently observed to inhibit Urokinase receptor expression, andsuccessfully inhibit colon cancer cell metastasis in vivo (Itoh et al.,20002, Abstract 409, Ann Meeting Am Soc Gen Ther www.asgt.org). Inanother application, DNAzymes complementary to bcr-ab1 oncogenes weresuccessful in inhibiting the oncogenes expression in leukemia cells, andlessening relapse rates in autologous bone marrow transplant in cases ofChronic Myelogenous Leukemia (CML) and Acute Lymphocytic Leukemia (ALL).

Another agent capable of downregulating H19RNA is a ribozyme moleculecapable of specifically cleaving its encoding polynucleotide. Ribozymesare being increasingly used for the sequence-specific inhibition of geneexpression by the cleavage of mRNAs encoding proteins of interest [Welchet al., Curr Opin Biotechnol. 9:486-96 (1998)]. The possibility ofdesigning ribozymes to cleave any specific target RNA has rendered themvaluable tools in both basic research and therapeutic applications. Inthe therapeutics area, ribozymes have been exploited to target viralRNAs in infectious diseases, dominant oncogenes in cancers and specificsomatic mutations in genetic disorders [Welch et al., Clin Diagn Virol.10:163-71 (1998)]. Most notably, several ribozyme gene therapy protocolsfor HIV patients are already in Phase 1 trials. More recently, ribozymeshave been used for transgenic animal research, gene target validationand pathway elucidation. Several ribozymes are in various stages ofclinical trials. ANGIOZYME was the first chemically synthesized ribozymeto be studied in human clinical trials. ANGIOZYME specifically inhibitsformation of the VEGF-r (Vascular Endothelial Growth Factor receptor), akey component in the angiogenesis pathway. Ribozyme Pharmaceuticals,Inc., as well as other firms have demonstrated the importance ofanti-angiogenesis therapeutics in animal models. HEPTAZYME, a ribozymedesigned to selectively destroy Hepatitis C Virus (HCV) RNA, was foundeffective in decreasing Hepatitis C viral RNA in cell culture assays(Ribozyme Pharmaceuticals, Incorporated—http://www.rpi.com/index.html).

An additional method of downregulating H19RNA is via triplex formingoligonucleotides (TFOs). In the last decade, studies have shown thatTFOs can be designed which can recognize and bind topolypurine/polypirimidine regions in double-stranded helical DNA in asequence-specific manner. Thus the DNA sequence encoding the H19 RNA ofthe present invention can be targeted thereby down-regulating the RNAmolecule.

The recognition rules governing TFOs are outlined by Maher III, L. J.,et al., Science (1989) 245:725-730; Moser, H. E., et al., Science (1987)238:645-630; Beal, P. A., et al., Science (1991) 251:1360-1363; Cooney,M., et al., Science (1988) 241:456-459; and Hogan, M. E., et al., EPPublication 375408. Modification of the oligonucleotides, such as theintroduction of intercalators and backbone substitutions, andoptimization of binding conditions (pH and cation concentration) haveaided in overcoming inherent obstacles to TFO activity such as chargerepulsion and instability, and it was recently shown that syntheticoligonucleotides can be targeted to specific sequences (for a recentreview see Seidman and Glazer (2003) J Clin Invest; 112:487-94).

In general, the triplex-forming oligonucleotide has the sequencecorrespondence:

oligo 3′--A G G T duplex 5′--A G C T duplex 3′--T C G AHowever, it has been shown that the A-AT and G-GC triplets have thegreatest triple helical stability (Reither and Jeltsch (2002), BMCBiochem, Sept12, Epub). The same authors have demonstrated that TFOsdesigned according to the A-AT and G-GC rule do not form non-specifictriplexes, indicating that the triplex formation is indeed sequencespecific.

Thus for any given sequence in the regulatory region a triplex formingsequence may be devised. Triplex-forming oligonucleotides preferably areat least 15, more preferably 25, still more preferably 30 or morenucleotides in length, up to 50 or 100 bp.

Transfection of cells (for example, via cationic liposomes) with TFOs,and subsequent formation of the triple helical structure with the targetDNA, induces steric and functional changes, blocking transcriptioninitiation and elongation, allowing the introduction of desired sequencechanges in the endogenous DNA and results in the specific downregulationof gene expression. Examples of such suppression of gene expression incells treated with TFOs include knockout of episomal supFG1 andendogenous HPRT genes in mammalian cells (Vasquez et al., Nucl AcidsRes. (1999) 27:1176-81, and Puri, et al., J Biol Chem, (2001)276:28991-98), and the sequence- and target-specific downregulation ofexpression of the Ets2 transcription factor, important in prostatecancer etiology (Carbone, et al., Nucl Acid Res. (2003) 31:833-43), andthe pro-inflammatory ICAM-1 gene (Besch et al., J Biol Chem, (2002)277:32473-79). In addition, Vuyisich and Beal have recently shown thatsequence specific TFOs can bind to dsRNA, inhibiting activity ofdsRNA-dependent enzymes such as RNA-dependent kinases (Vuyisich andBeal, Nuc. Acids Res (2000); 28:2369-74).

Additionally, TFOs designed according to the abovementioned principlescan induce directed mutagenesis capable of effecting DNA repair, thusproviding both downregulation and upregulation of expression ofendogenous genes [Seidman and Glazer, J Clin Invest (2003) 112:487-94].Detailed description of the design, synthesis and administration ofeffective TFOs can be found in U.S. Patent Application Nos. 2003 017068and 2003 0096980 to Froehler et al., and 2002 0128218 and 2002 0123476to Emanuele et al., and U.S. Pat. No. 5,721,138 to Lawn.

It will be appreciated that nucleic acid agents capable of hybridizingH19 mRNA may down-regulate an activity thereof by preventing H19 mRNAbinding to another downstream agent.

As mentioned hereinabove, the nucleic acid agents of the presentinvention (e.g., an siRNA molecule such as those set forth by SEQ IDNO:1, 2, 3 or 4) can be expressed in cells.

It will be appreciated that the agents of the present invention may beexpressed directly in the subject (i.e. in vivo gene therapy) or may beexpressed ex vivo in a cell system (autologous or non-autologous) andthen administered to the subject.

To express such an agent (i.e., to produce an RNA molecule) in mammaliancells, a nucleic acid sequence encoding the agents of the presentinvention is preferably ligated into a nucleic acid construct suitablefor mammalian cell expression. Such a nucleic acid construct includes apromoter sequence for directing transcription of the polynucleotidesequence in the cell in a constitutive or inducible manner.

Constitutive promoters suitable for use with the present invention arepromoter sequences which are active under most environmental conditionsand most types of cells such as the cytomegalovirus (CMV) and Roussarcoma virus (RSV). Inducible promoters suitable for use with thepresent invention include for example the tetracycline-induciblepromoter (Zabala M, et al., Cancer Res. 2004, 64(8): 2799-804).

The nucleic acid construct (also referred to herein as an “expressionvector”) of the present invention includes additional sequences whichrender this vector suitable for replication and integration inprokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). Inaddition, typical cloning vectors may also contain a transcription andtranslation initiation sequence, transcription and translationterminator and a polyadenylation signal.

Eukaryotic promoters typically contain two types of recognitionsequences, the TATA box and upstream promoter elements. The TATA box,located 25-30 base pairs upstream of the transcription initiation site,is thought to be involved in directing RNA polymerase to begin RNAsynthesis. The other upstream promoter elements determine the rate atwhich transcription is initiated.

Preferably, the promoter utilized by the nucleic acid construct of thepresent invention is active in the specific cell population transformed.Examples of cell type-specific and/or tissue-specific promoters includepromoters such as albumin that is liver specific [Pinkert et al., (1987)Genes Dev. 1:268-277], lymphoid specific promoters [Calame et al.,(1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cellreceptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins;[Banerji et al. (1983) Cell 33729-740], neuron-specific promoters suchas the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad.Sci. USA 86:5473-5477], pancreas-specific promoters [Edlunch et al.(1985) Science 230:912-916] or mammary gland-specific promoters such asthe milk whey promoter (U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166).

Enhancer elements can stimulate transcription up to 1,000 fold fromlinked homologous or heterologous promoters. Enhancers are active whenplaced downstream or upstream from the transcription initiation site.Many enhancer elements derived from viruses have a broad host range andare active in a variety of tissues. For example, the SV40 early geneenhancer is suitable for many cell types. Other enhancer/promotercombinations that are suitable for the present invention include thosederived from polyoma virus, human or murine cytomegalovirus (CMV), thelong term repeat from various retroviruses such as murine leukemiavirus, murine or Rous sarcoma virus and HIV. See, Enhancers andEukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor,N.Y. 1983, which is incorporated herein by reference.

In the construction of the expression vector, the promoter is preferablypositioned approximately the same distance from the heterologoustranscription start site as it is from the transcription start site inits natural setting. As is known in the art, however, some variation inthis distance can be accommodated without loss of promoter function.

Polyadenylation sequences can also be added to the expression vector inorder to increase RNA stability [Soreq et al., 1974; J. Mol. Biol. 88:233-45).

Two distinct sequence elements are required for accurate and efficientpolyadenylation: GU or U rich sequences located downstream from thepolyadenylation site and a highly conserved sequence of six nucleotides,AAUAAA, located 11-30 nucleotides upstream. Termination andpolyadenylation signals that are suitable for the present inventioninclude those derived from SV40.

In addition to the elements already described, the expression vector ofthe present invention may typically contain other specialized elementsintended to increase the level of expression of cloned nucleic acids orto facilitate the identification of cells that carry the recombinantDNA. For example, a number of animal viruses contain DNA sequences thatpromote the extra chromosomal replication of the viral genome inpermissive cell types. Plasmids bearing these viral replicons arereplicated episomally as long as the appropriate factors are provided bygenes either carried on the plasmid or with the genome of the host cell.

The vector may or may not include a eukaryotic replicon. If a eukaryoticreplicon is present, then the vector is amplifiable in eukaryotic cellsusing the appropriate selectable marker. If the vector does not comprisea eukaryotic replicon, no episomal amplification is possible. Instead,the recombinant DNA integrates into the genome of the engineered cell,where the promoter directs expression of the desired nucleic acid.

Examples for mammalian expression vectors include, but are not limitedto, pcDNA3, pcDNA3.1 (+/−), pGL3, pZeoSV2(+/−), pSecTag2, pDisplay,pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1,pNMT41, pNMT81, which are available from Invitrogen, pCI which isavailable from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which areavailable from Strategene, pTRES which is available from Clontech, andtheir derivatives.

Expression vectors containing regulatory elements from eukaryoticviruses such as retroviruses can be also used. SV40 vectors includepSVT7 and pMT2. Vectors derived from bovine papilloma virus includepBV-1MTHA, and vectors derived from Epstein Bar virus include pHEBO, andp2O5. Other exemplary vectors include pMSG, pAV009/A⁺, pMTO10/A⁺,pMAMneo-5, baculovirus pDSVE, and any other vector allowing expressionof proteins under the direction of the SV-40 early promoter, SV-40 laterpromoter, metallothionein promoter, murine mammary tumor virus promoter,Rous sarcoma virus promoter, polyhedrin promoter, or other promotersshown effective for expression in eukaryotic cells.

As described above, viruses are very specialized infectious agents thathave evolved, in many cases, to elude host defense mechanisms.Typically, viruses infect and propagate in specific cell types. Thetargeting specificity of viral vectors utilizes its natural specificityto specifically target predetermined cell types and thereby introduce arecombinant gene into the infected cell. Thus, the type of vector usedby the present invention will depend on the cell type transformed. Theability to select suitable vectors according to the cell typetransformed is well within the capabilities of the ordinary skilledartisan and as such no general description of selection consideration isprovided herein. For example, bone marrow cells can be targeted usingthe human T cell leukemia virus type I (HTLV-I) and kidney cells may betargeted using the heterologous promoter present in the baculovirusAutographa californica nucleopolyhedrovirus (AcMNPV) as described inLiang C Y et al., 2004 (Arch Virol. 149: 51-60).

Recombinant viral vectors are useful for in vivo expression of the H19down-regulating agents of the present invention since they offeradvantages such as lateral infection and targeting specificity. Lateralinfection is inherent in the life cycle of, for example, retrovirus andis the process by which a single infected cell produces many progenyvirions that bud off and infect neighboring cells. The result is that alarge area becomes rapidly infected, most of which was not initiallyinfected by the original viral particles. This is in contrast tovertical-type of infection in which the infectious agent spreads onlythrough daughter progeny. Viral vectors can also be produced that areunable to spread laterally. This characteristic can be useful if thedesired purpose is to introduce a specified gene into only a localizednumber of targeted cells.

Various methods can be used to introduce the expression vector of thepresent invention into cells. Such methods are generally described inSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringsHarbor Laboratory, New York (1989, 1992), in Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich.(1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995),Vectors: A Survey of Molecular Cloning Vectors and Their Uses,Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4(6): 504-512, 1986] and include, for example, stable or transienttransfection, lipofection, electroporation and infection withrecombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and5,487,992 for positive-negative selection methods.

Introduction of nucleic acids by viral infection offers severaladvantages over other methods such as lipofection and electroporation,since higher transfection efficiency can be obtained due to theinfectious nature of viruses.

Currently preferred in vivo nucleic acid transfer techniques includetransfection with viral or non-viral constructs, such as adenovirus,lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) andlipid-based systems. Useful lipids for lipid-mediated transfer of thegene are, for example, DOTMA, DOPE, and DC-Chol [Tonkinson et al.,Cancer Investigation, 14(1): 54-65 (1996)]. The most preferredconstructs for use in gene therapy are viruses, most preferablyadenoviruses, AAV, lentiviruses, or retroviruses. A viral construct suchas a retroviral construct includes at least one transcriptionalpromoter/enhancer or locus-defining element(s), or other elements thatcontrol gene expression by other means such as alternate splicing,nuclear RNA export, or post-translational modification of messenger.Such vector constructs also include a packaging signal, long terminalrepeats (LTRs) or portions thereof, and positive and negative strandprimer binding sites appropriate to the virus used, unless it is alreadypresent in the viral construct. Optionally, the construct may alsoinclude a signal that directs polyadenylation, as well as one or morerestriction sites. By way of example, such constructs will typicallyinclude a 5′ LTR, a tRNA binding site, a packaging signal, an origin ofsecond-strand DNA synthesis, and a 3′ LTR or a portion thereof. Othervectors can be used that are non-viral, such as cationic lipids,polylysine, and dendrimers.

Other than containing the necessary elements for the transcription ofthe inserted coding sequence, the expression construct of the presentinvention can also include sequences engineered to enhance stability,production, purification, yield or toxicity of the expressed RNA.

As mentioned above, agents capable of down-regulating H19 mRNA can beused to treat cancer either alone (e.g. siRNAs of the present invention)or in combination with other established or experimental therapeuticregimen for such disorders. The present inventors envisage that agentscapable of down-regulating H19 mRNA may act synergistically withadditional therapeutic methods or compositions and therefore have thepotential to significantly reduce the effective clinical doses of suchtreatments, thereby reducing the often devastating negative side effectsand high cost of the treatment. This may be particularly relevant fortreating solid tumors associated with hypoxic regions wherebyestablished chemotherapy and radiotherapy regimens are ineffective.

Agents of the present invention may be administered prior to,concommitedly or following the cancer therapy.

As used herein the phrase “cancer therapy” refers to any treatment whichacts to prevent, alleviate or diminish a symptom associated with acancerous disease.

Therapeutic regimen for treatment of cancer suitable for combinationwith the agents of the present invention or polynucleotide encoding sameinclude, but are not limited to chemotherapy, radiotherapy, phototherapyand photodynamic therapy, surgery, nutritional therapy, ablativetherapy, combined radiotherapy and chemotherapy, brachiotherapy, protonbeam therapy, immunotherapy, cellular therapy and photon beamradiosurgical therapy. Another form of therapeutic regimen for treatmentof cancer suitable for combination with the agents of the presentinvention is the administration of nucleotide agents which are capableof regulating genes known to be involved in a cancer-regulating orangiogenesis-regulating pathway.

Anti-cancer drugs (i.e. chemotherapeutic agents) that can beco-administered with the compounds of the invention include, but are notlimited to Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine;Adriamycin; Adozelesin; Aldesleukin; Altretamine; Ambomycin; AmetantroneAcetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin;Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat;Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate;Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan;Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin;Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol;Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate;Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; DaunorubicinHydrochloride; Decitabine; Dexormaplatin; Dezaguanine; DezaguanineMesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride;Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin;Edatrexate; Eflornithine Hydrochloride; Elsamitrucin; Enloplatin;Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole;Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium;Etanidazole; Etoposide; Etoposide Phosphate; Etoprine; FadrozoleHydrochloride; Fazarabine; Fenretinide; Floxuridine; FludarabinePhosphate; Fluorouracil; Fluorocitabine; Fosquidone; Fostriecin Sodium;Gemcitabine; Gemcitabine Hydrochloride; Hydroxyurea; IdarubicinHydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; InterferonAlfa-2b; Interferon Alfa-n1; Interferon Alfa-n3; Interferon Beta-I a;Interferon Gamma-I b; Iproplatin; Irinotecan Hydrochloride; LanreotideAcetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride;Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol;Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate;Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine;Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide;Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper;Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole;Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin;Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan;Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium;Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin;Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol;Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium;Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin;Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Taxol; TecogalanSodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide;Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa;Tiazofuirin; Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate;Trestolone Acetate; Triciribine Phosphate; Trimetrexate; TrimetrexateGlucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard;Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate; VincristineSulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; VinglycinateSulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; VinrosidineSulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin;Zorubicin Hydrochloride. Additional antineoplastic agents include thosedisclosed in Chapter 52, Antineoplastic Agents (Paul Calabresi and BruceA. Chabner), and the introduction thereto, 1202-1263, of Goodman andGilman's “The Pharmacological Basis of Therapeutics”, Eighth Edition,1990, McGraw-Hill, Inc. (Health Professions Division).

The agents of the present invention may, if desired, be presented in apack or dispenser device, such as a FDA approved kit, which may containone or more unit dosage forms containing the agents of the presentinvention. The agents may be co-formulated in a single packaging withthe additional anti cancer agent or the agents may be formulatedseparately from the additional anti-cancer agent in separate packaging.The pack may, for example, comprise metal or plastic foil, such as ablister pack. The pack or dispenser device may be accompanied byinstructions for administration. The pack or dispenser may also beaccompanied by a notice associated with the container in a formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the compositions or human or veterinaryadministration. Such notice, for example, may be of labeling approved bythe U.S. Food and Drug Administration for prescription drugs or of anapproved product insert. Compositions comprising the agents of theinvention formulated in a compatible pharmaceutical carrier may also beprepared, placed in an appropriate container, and labeled for thetreatment of cancer.

The agents of the present invention can be administered to a subject perse, or in a pharmaceutical composition where it is mixed with suitablecarriers or excipients.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the agent accountable forthe anti-cancer effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into the tumor (i.e. in situ).

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients (nucleic acid construct) effective to prevent,alleviate or ameliorate symptoms of a disorder (e.g., ischemia) orprolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin animal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provideplasma or brain levels of the active ingredient are sufficient to induceor suppress the biological effect (minimal effective concentration,MEC). The MEC will vary for each preparation, but can be estimated fromin vitro data. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. Detection assayscan be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as if further detailed above.

It is expected that during the life of this patent many relevant cancertherapies will be developed and the scope of the term cancer therapy isintended to include all such new technologies a priori.

As used herein the term “about” refers to ±10%.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Example 1 Detection of an Alternative Splice Variant of H19 in HumanEmbryonic and Placental Specimens

The following experiments were performed in order to ascertain whethersplice variants of H19 were restricted to a particular cell type.

Materials and Methods

Cell culture: All the human carcinoma cell lines used in this study wereobtained from the American type culture collection (Manassas, Va.) andwere maintained in DMEM-F12 (1:1) medium containing 10% fetal calf serum(inactivated 55° C. for 30 minutes), 25 mM HEPES (pH 7.4), penicillin(180 units/ml), streptomycin (100 μg/ml) and amphotericin B (0.2 μg/ml).Approximately 4×10⁴ cells/cm² were plated in polystyrene culture dishes(NUNC). Every 4 days, the cells were trypsinized with 0.05% trypsin-EDTAsolution (Biet Haemek) for 10 minutes and re-plated again at the sameinitial densities.

Reverse Transcriptase Polymerase Chain Reaction (RT-PCR): Total RNA wasextracted from tissues and cultured cell lines using the TRI REAGENT(Sigma) according to the manufacturer's instructions and treated withDNase I to exclude genomic DNA contamination as described previously(Ayesh and Matouk et al, 2002, Mol Ther 7, 535-541). The synthesis ofcDNA was performed using the p(dT)15 primer (Roche, Germany), toinitiate reverse transcription of 5 μg total RNA with 400 units ofReverse Transcriptase (Gibco BRL), according to manufacturer'sinstructions. The PCR reaction was carried out in the presence of DiazadGTP (Roche, Germany) with Taq polymerase (Takara, Otsu, Japan) for 40cycles (94° C. for 1 min, 58° C. for 30 s, and 72° C. for 40 s) precededby 94° C. for 5 min, and a final extension of 5 min at 72° C. Theprimers used in the PCR reaction were (5′-AGGAGCACCTTGGACATCTG-3′) (SEQID NO: 8) and (5′-CCCCTGTGCCTGCTACTAAA-3′) (SEQ ID NO: 9) and were 117and 816 bases downstream to the published transcription H19 initiationsite, respectively (Brannan et al, 1990, Mol Cell Biol 10, 28-36). Theposition of the primers is illustrated in FIG. 1A. The products of thePCR reaction were run on ethidium bromide stained gels.

Probe Synthesis: PCR products from tissues demonstrating the minor bandwere purified from the gel by the GFX™ PCR, DNA and Gel BandPurification Kit, and cloned into a T-easy® vector (Promega, USA). Theorientation of the insert was verified by restriction enzyme analysis,and accordingly the labeled antisense strand was synthesized usingDigoxigenin UTP, according to the supplier's instructions (Roche,Germany). The resulting probe was treated with 2 units of RNase freeDNase I, pelleted and resuspended in an appropriate volume ofDEPC-treated double distilled water. The size of the synthesized probewas analyzed by running on a 4% denaturing agarose minigel, and itslabeling efficiency was determined by dot blot analysis usingDigoxigenin antibody (data not shown).

RNase Protection Assay: Various concentrations of third trimesterplacenta RNA (which showed the presence of the alternative splicevariant using the RT-PCR assay) were used in an RNase protection assay.600 pg Dig-labeled probe/10 μg total RNA (DNase I treated) from thirdtrimester placenta and yeast RNA equals to the highest concentration ofRNA used were hybridized at 42° C. for 16 hours and digested with RNaseA/and RNase TI, according to the kit instructions RPA II™ (Ambion). TheRNA fragments protected from RNase digestion were separated byelectrophoresis on a 5% polyacrylamide gel (containing 8 M urea) andwere detected using the CDP Star Detection Kit (Roche, Germany),according to the manufacturer's instructions.

DNA Sequencing: Sequencing reactions were carried out using the ABIPRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE AppliedBiosystem).

Results

An alternatively spliced variant of H19 was present in placental andembryonic tissues and not in carcinoma cell lines, and cancer patientspecimens as demonstrated by RT-PCR analysis (FIGS. 1B-D) and RNaseprotection assay (FIG. 1E). Sequencing studies showed that thealternative spliced variant was 344 bp long and lacked part of exon-1extending from nt 252 to 588 of the transcription start site (FIG. 1F)as compared to the known H19 transcript (GenBank Accession No. M32053).

Example 2 H19 Gene Expression is Moderately Up-Regulated by CoCl₂

Several genes up regulated in the presence of H19 RNA are also known tobe induced by hypoxia (Ayesh and Matouk et al, 2002, Mol Carcinog 35,63-74). It was also reported that H19 RNA has been detected inrheumatoid arthritis synovial tissue (Stuhlmuller et al, 2003, Am JPathol 163, 901-911). The presence of extensive angiogenesis is usuallyassociated with rheumatoid arthritis due to hypoxic and oxidativestress, partly due to the metabolic activity of increased inflammatorycell exudates in the affected area.

Moreover, a proteomic approach has revealed that H19 over-expression inhuman cancerous mammary epithelial cells stably transfected with genomicDNA containing the entire H19 gene, is responsible for positivelyregulating the thioredoxin gene at post-transcriptional level,thioredoxin being a key protein of the oxidative stress response anddeoxynucleotide biosynthesis (Lottin et al, 2002, Carcinogesis 23,1885-1895).

In addition, many processes that involve cellular invasion, includingblastocyst implantation, and placental development occur in reducedoxygen environments (Rodesch et al, 1992, Obstet Gynecol 80, 283-285).These two physiological processes show intensive up regulation of H19expression (Ariel et al, 1994, Gynecol Oncol 53, 212-219).

Based on those reasoning, the H19 gene was analyzed to determine if itwas sensitive to hypoxia.

Materials and Methods

Hep3B cells were cultured in normal medium conditions for 24 hours priorto CoCl₂ manipulation. The cells were incubated with CoCl₂ (Sigma,Aldrich) for a further 22 hours prior to RNA extraction.

RT-PCR analysis was performed as described above in Example 1 using thefollowing primers 5′-CCG GCC TTC CTG AAC A-3′ Forward (SEQ ID NO: 10)and 5′-TTC CGA TGG TGT CTT TGA TGT-3′ Reverse (SEQ ID NO: 11)

Results

H19 gene expression is moderately upregulated in Hep3B cells (FIGS.2A-B) in response to the addition of increasing concentrations of CoCl₂(50-400 uM) as tested by RT-PCR analysis. This moderate up-regulationrelative to the strong up-regulation towards real hypoxic conditionsindicate that HIF-α is only partly responsible.

Example 3 H19 RNA is Efficiently Down-Regulated In Vitro by DifferentsiRNA Duplexes in Both Normal and Hypoxia-Like Culture Conditions

Materials and Methods

siRNAs preparation: Four siRNAs targeting human H19 and one negativecontrol siRNA (targeting luciferase pGL3) (as set forth in Table 1hereinbelow) were synthesized as a ready to use duplexes by Proligo anddesigned as recommended with dTdT 3′ overhangs on each strand. Allsequences were evaluated for gene specificity using the NationalInstitutes of Health Blast program.

TABLE 1 siRNA SEQ ID name Sense sequence Location NO: H19 siRNA-15′-UAAGUCAUUUGCACUGGUUdTdT-3′ Exon 5 1 H19 siRNA-25′-GCAGGACAUGACAUGGUCCdTdT-3′ Exon 2 2 H19 siRNA-35′-CCAACAUCAAAGACACCAUdTdT-3′ Exon 5 3 H19 siRNA-45′-CCAGGCAGAAAGAGCAAGAdTdT-3′ Exon 1 4 PG13 siRNA5′-CUUACGCUGAGUACUUCGAdTdT-3′ Exon 1 5 GFP siRNA5′- GCA AGC UGA CCC UGA AGU UCA U 6

Upon receiving each freeze dried siRNA was reconstituted with RNase freewater to prepare a 50 pmole/ul solution and stored as aliquots at −80°C.

Cell culture conditions and transfection of siRNAs: Transfection ofsiRNAs was conducted with lipofectamine 2000 (Invitrogen, US) in 12-wellplates. The day prior to transfection, the cells were trypsinized,counted, and seeded at 60,000/well containing 1 ml DMEM medium withoutantibiotics so that they were nearly 50% confluent on the day oftransfection. 3 μl of lipofectamine 2000 was incubated for 15 minuteswith 100 μl serum-free OPTI-MEM medium, (Invitrogen, US). This was addedto the 100 pmole dsRNA diluted in 100 μl serum free OPTI-MEM media andthe formulation lasted 20 minutes. 195 μl of the mixture was applied toHep3B cells and UMUC3 cells and incubated for another 48 hours withoutreplacement of the medium. For hypoxia mimicking conditions freshlyprepared CoCl₂ was added at a final concentration of 100 μM 24 hourspost transfection and the cells were incubated for a further 22 hoursprior to RNA extraction.

RNA extraction and RT-PCR conditions (siRNA): Total RNA and reversetranscription was performed as described above in Example 1 except that1 μg of total RNA was used. The PCR reaction for H19 was carried out inthe presence of Taq polymerase (Takara, Otsu, Japan) for 34 cycles (94°C. for 30 s, 58° C. for 30 s, and 72° C. for 30 s) preceded by 94° C.for 5 min, and a final extension of 5 min at 72° C., and for GADPH andhistone. Primer sequences for GAP: forward 5′-GGC TCT CCA GAA CAT CATCCC TGC-3′ (SEQ ID NO: 12) and Reverse GGG TGT CGC TGT TGA AGT CAGAGG-3′ (SEQ ID NO: 13).

Results

Hep3B cells: The ability of siRNA to reduce the endogenous level of H19RNA under both normal (FIG. 3A), or hypoxia like conditions (FIG. 3B)was examined. Dramatic suppression of H19 expression was detected byRT-PCR analysis (48 hours post transfection) using four different siRNAs(1-4) targeting H19 (FIG. 3A, lanes 2-5) or the equimolar pool of thefour siRNA (FIG. 3A, lane 6) but not with non-related PGl3 duplextargeting luciferase (FIG. 3A, lane 1) and mock (FIG. 3A, lane 7)respectively. Moreover, the ability of three different siRNA (1, 3 and4) to suppress the expression of H19 gene was tested in hypoxia-likeCoCl₂ simulation (FIGS. 3B-C). While H19 RNA is moderately induced byCoCl₂ simulation (compare FIG. 3B lanes 1 for hypoxic simulation and 5for normal both transfected with PGl3 duplexes), dramatic reduction wasdetected using three different siRNA targeting H19 transcript (FIG. 3B,lanes 2-4).

UMUC3 cells: As with Hep3B cells, hypoxic conditions increased theexpression of H19 message and the siRNA H19 (SEQ ID NO: 1) verysignificantly reduced its expression (FIGS. 3D-E).

Example 4 Ex-Vivo Down-Regulation of H19 RNA in Both Hep3B and UMUC3Cells Reduces In Vivo Tumorigenicity

Materials and Methods

Ex-vivo tumorigenic assay: Hep3B and UMUC3 cells were transfected invitro by two different siRNA duplexes directed against H19 RNA (siRNASEQ ID NO: 3 for Hep3B cells and siRNA SEQ ID NO: 1 for UMUC3) and anunrelated control siRNA (targeting Luc or GFP), respectively asdescribed above. Forty eight hours post transfection, cells wereinjected subcutaneously into the dorsal flank region of athymic nudemice. An additional control group was without any treatment. Cells weretrypsinized, counted, and centrifuged and re-suspended into sterile PBS(1×), so that there were about 5×10⁶ cells/ml. 250 μl of the suspensionwas injected into the dorsal flank region of athymic nude mice. Fifteenand 30 days post injection, tumors begin to develop and their volumeswere measured using a caliper.

Transfection of siRNAs was conducted with lipofectamine 2000(Invitrogen, US) in 6-wells plates. The day prior to transfection, thecells were trypsinized, counted, and seeded at 100,000/well containing 2ml DMEM medium without antibiotics so that they were nearly 50%confluent on the day of transfection. 5 μl of lipofectamine 2000incubated for 15 minutes with 250 μl serum-free OPTI-MEM medium(Invitrogen, US). This was added to the 100 uM dsRNA diluted in 250 μlserum free OPTI-MEM media and the formulation lasted 20 minutes. 500 μlof the mixture was applied to the cells and incubated for another 48hours without replacement of the medium. Each treatment group comprisedseven mice.

Results

As illustrated in FIGS. 4A-D, administration of Hep3B cells previouslytransfected with H19 siRNA to mice caused a very significant lowering intumor weight (FIG. 4A) and volume (FIG. 4B) than Hep3B cells transfectedwith Luc siRNA. As illustrated in FIGS. 5A-D, UMUC3 cells transfectedwith H19 siRNA also caused a very significant lowering in tumor weight(FIG. 5A) and volume (FIG. 5B) in mice than UMUC3 cells transfected withLuc siRNA.

Example 5 Oncogenic Properties of H19 siRNA

In order to ascertain whether H19 RNA is a tumor-associated gene productor whether it is potentially harboring an oncogenic potential by itself,the following experiment was performed.

Materials and Methods

Cell proliferation analysis: Hep3B cells were seeded and transfected in12 well plates with anti-Luc siRNA or H19 siRNA (SEQ ID NO: 3). Twentyfour hours later, cells were washed twice with PBS, trypsinized andcounted. 5×10³ transfected Hep3B cells were seeded in quadruples in 96well plates in DMEM media containing 10% FCS, and further incubated for24 hours before the MTS assay was performed. MTS assay was performedaccording to the procedure provided by the supplier (Promega, USA). Theabsorbance at 940 nm was recorded using ELISA plate reader.

Results

As shown in FIG. 6, siRNA H19 did not induce a statistically significantattenuation of cell proliferation of Hep3B cells.

Moreover the effect of H19 suppression on anchorage independent colonyformation in soft agar after hypoxia recovery was also analyzed as anadditional assessment of tumorigenicity in vitro. Hep3B cells wereexposed to hypoxic stress 4 hours post transfection as described in thematerials and methods. 24 hours post hypoxic conditions, cells wereseeded on soft agar. H19 siRNA significantly abrogated anchorageindependent growth after hypoxia recovery in which both colony numberand size were very significantly reduced (FIG. 3F).

Example 6 In-Vivo Intra-Tumoral Injection of H19 siRNA Duplex

Materials and Methods

Preparation of H19siRNAs: The transfectant used was jetPEI™ (×4) concfrom Polyplus. 850 pmoles (˜11 μg of siRNAs), and 10 μl of jetPEI(N/P=10), were diluted in 100 μl 5% glucose solution, 5 minutes after,jetPEI solution was added to siRNAs solutions and the formulation lasted20 minutes before intratumoral (for UMUC3) or initial inoculation site(for Hep3B) injections.

Experimental procedure: 2×10⁶ bladder carcinoma cells (UMUC3), andhepatocellular carcinoma (Hep3B) cells were suspended in 100 μl PBS andinjected subcutaneously in the dorsa of 10 athymic male mice, for UMUC3and 8 for Hep3B.

UMUC3 cells: When the tumors reach about 4-8 mm in diameter in UMUC3,mice were segregated to two homogeneous groups (n=5), and received thefirst intratumoral siRNA injection of unrelated GFP as a control or H19siRNAs (H19 siRNA-3—SEQ ID NO: 3). A total of 3 injections wereadministered at 2 and 5 days intervals following the first intratumoralinjection and mice were left 6 days post final injection without anytreatment. Tumor volumes for the two treated groups were measured usinga caliper, and their final tumor weights were recorded.

Hep3B cells: For Hep3B cells, treatment followed 48 hours following cellinoculation before palpable tumors were observed. The mice weresegregated into two groups (n=4 each), and injected at the site ofinitial inoculation. Mice received a total of 5 injections, every twodays, and then left for a week post final injection before scarifyingthem.

Tumor volume was calculated by the equation, V=(L×W²)×0.5 (V, volume; L,length; and W, width).

Results

To determine the functional consequences of H19 knockdown in tumorgrowth, H19 siRNA-PEI complex was injected into small tumors inducedfrom bladder carcinoma UMUC3 cell line and before palpable tumors wereobserved in Hep3B carcinoma cell line in nude mice. Synthetic controlsiRNA targeting GFP formulated with PEI was used as a control. As shownin FIGS. 7A-B, H19 siRNA3 causes a very significant reduction of about90% of mean tumor volumes (FIG. 7A), and of about 88% of mean tumorweights (FIG. 7B) in UMUC3 cells.

In Hep3B induced tumors, the level of reduction in tumor volumes andweights are less pronounced using siRNA1. An approximate 40% reductionof tumor weights (FIG. 7C) and 56% reduction of tumor volumes (FIG. 7D)were observed.

Example 7 H19 Involvement in TA11 and TA31 Cells

Two human bladder carcinoma cells lines, TA11 and TA31, originating fromthe same parental cell line T24P were shown to be either negative,(TA11H19-ve) or to be high expressers (TA31H19high) of H19 in vitrounder normal culture conditions, respectively [Ayesh et al, 2002, MolCarcinog 35, 63-74]. The following experiment was performed in order todetermine whether the H19 message effects tumor growth of these othercell lineages.

Materials and Methods

TA11 and TA31 cells (approximately 2×10⁶) were implanted subcutaneouslyinto CD-1 mice (n=5 each). Tumor volumes were measured 15 dayspost-implantation. As shown in FIGS. 8A-B, tumors derived from theTA11H19-ve cells were significantly smaller than those from theTA31H19high cells. Furthermore, the TA31H19high-derived tumors weresignificantly more vascularized (FIGS. 8C-D). RT-PCR results from thetumors that obtained from TA11H19-ve cells show that H19 RNA is inducedin those tumors as opposed to null expression of H19 RNA in those cellsin vitro (data not shown). These results suggest that H19 RNA enhancestumor growth.

Example 8

H19 RNA is Induced by Hypoxic Stress in Hep3B Cell Line and siRNADirected Against H19 Very Efficiently Impedes its Induction

Materials and Methods

Hep3B cells were seeded and transfected either with anti H19 siRNA oranti Luc siRNA as described above. 24 hours post transfection, cellswere either placed into Aneoropack rectangular jar (Mitsubishi chemicalcompany Japan) to create a hypoxic conditions within an hour (1% O₂, 20%CO₂), or left in normal oxygen concentrations. Incubation lasted for 24hours prior to RNA extraction. RT-PCR analysis was performed asdescribed for Example 1 hereinabove.

Results

As illustrated in FIG. 9A, H19 RNA was specifically down-regulated bothin normal (FIG. 9A, lane 3) and hypoxic (FIG. 9A, lane 4) cultureconditions respectively. PCR analysis of house-keeping genes (GAPDH) isillustrated in FIG. 9B. PCR analysis of uPAR is illustrated in FIG. 9C.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

1. An isolated oligonucleotide able to down-regulate H19 mRNA under bothnormal and hypoxic conditions as set forth in SEQ ID NO:
 1. 2. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and, as an active ingredient, at least one isolatedoligonucleotide according to claim
 1. 3. An article of manufactureidentified for treating cancer comprising an agent capable ofdownregulating a level and/or activity of H19 mRNA, wherein said agentis an siRNA able to down-regulate H19 mRNA under both normal and hypoxicconditions which comprises a nucleic acid sequence selected from thegroup consisting of as set forth in SEQ ID NO: 1, and an additional anticancer agent.
 4. The article of manufacture of claim 3, wherein saidcancer is a bladder carcinoma or a hepatocellular carcinoma.
 5. Thearticle of manufacture of claim 3, wherein said agent capable ofdownregulating a level and/or activity of H19 mRNA is co-formulated withsaid additional anti cancer agent.
 6. A nucleic acid construct encodingthe oligonucleotide of claim 1.